Scanning probe microscopes (SPMs) obtain data regarding surface topography by using a sharp tip or probe on the end of a cantilever held on or at a short distance (e.g., about 5-500 Å) from a sample. The cantilever tip can be deflected by various forces acting at the interface between the sample and the tip, such as electrostatic, magnetic, and van der Waals forces. A movement of the cantilever due to interactions between an atom at the end of the tip and an atom of the sample can then be measured electrically (as in a Scanning Tunneling Microscope, or STM) or optically (as in an Atomic Force Microscope, or AFM). By scanning the sample in x- and y-directions to change its position relative to the cantilever tip, three-dimensional information regarding the surface features of a sample can be obtained (see, e.g., Binnig, et al, 1986, Phys. Rev. Lett. 56(9): 930-933); McClelland, et al., 1987, Rev. Progr. Quart. Non-Destr. Eval. 6: 1307; Martin, et al, 1987, J. Appl. Phys. 61(10): 4723-4729).
AFM cantilevers operate in an oscillating mode or in a non-oscillating mode and can further interact with a sample in a contact mode or in a non-contact mode. In an oscillating contact mode, the cantilever is oscillated mechanically at or near its resonant frequency so that its tip repeatedly taps a sample surface, thus reducing the tip's oscillation amplitude. In an oscillating non-contact mode, interactions between the sample and the tip alter the tip's oscillation amplitude or frequency. The change in oscillation amplitude indicates proximity to the sample surface and may be used as a signal for feedback (e.g., for control of probe scanning). In a non-oscillating contact mode, the cantilever is not oscillated, and cantilever deflection is monitored as the tip is dragged over the sample surface, while in a non-oscillating non-contact mode, attractive interactions between the tip and the sample shift the cantilever resonance frequency.
Atomic force microscopy is emerging as an important tool in methods which rely on detecting information about surface features of a sample, measuring forces between two surfaces, or fabricating nanostructures (e.g., on silicon wafers, thin film magnetic read/write heads, and the like) (see, e.g., U.S. Pat. No. 6,337,479).
Atomic force microscopy also has many applications in biomedical research. It can be used for high contrast, high resolution imaging of biological surfaces in a wide range of environments (Engel, et al., 1999, Trends Cell Biol. 9: 77-80; Czajkowsky and Shao, 1998, FEBS Lett. 430: 51-4. 1998; Bustamante, et al., 1997, Curr. Opin. Struc. Biol. 7: 709-16; Hansma and Hoh, 1994, Ann. Rev. Biophys. Biomol. Struct. 23: 115-39.) It also can be used to measure intermolecular forces (e.g., Heinz and Hoh; 1999, Trends Biotech. 17:143-150; Mann, S. and H. E. Gaub, 1997, Curr. Opin. Colloid Interface Sci. 2: 145-152; Cappella and Dietler, 1999, Surf. Sci. Rep. 34: 1), intramolecular forces (Lee, et al., 1994; Lee, et al., 1994, Science 266: 771-773. 1994; Rief, et al., 1997, Science 275: 1295-7), and local mechanical properties (A-Hassan et al., 1998, Biophys. J. 74: 1564-1578; Vinckier and Semenza, 1998, FEBS Lett. 430: 12-6. 1998; Radmacher, et al., 1996, Biophys. J. 70: 556-567). Imaging with AFM offers advantages for studying biological samples, because the samples do not require drying, sectioning, metal coating or chemical fixing prior to analysis. Thus, AFMs may be used with samples that require very little sample preparation, including samples that are biologically active in both ambient air (including dried samples) and liquid.
One of the limiting elements in current AFMs is the design of the cantilever. The performance of the cantilever is primarily constrained by a combination of its fundamental resonant frequency (ω) and spring constant (k). Typical cantilevers of the prior art are on the order of 85-500 μm long and have resonant frequencies substantially less than 500 KHz. For example, generally, prior art cantilevers have lengths on the order of 85-320 μm, widths of 10-20 μm and thicknesses on the order of 0.5 μm. This produces typical [k, ωs] pairs of [0.5 N/m, 30 kHz] for shorter cantilevers, and [0.01 N/m, 2 kHz] for the longer cantilevers (where ωs is the fundamental resonant frequency in solution).
Smaller cantilevers with higher resonant frequencies are desirable because they allow faster imaging rates and permit the cantilever tip to more closely track sample topography (see, e.g., Butt, Biophys. J. 60: 777-785). Smaller cantilevers also are less affected by viscous damping, and are therefore more sensitive (see, e.g., U.S. Pat. No. 6,016,693). Smaller cantilevers have been described in Walters, et al., 1996, Rev. Sci. Instrum. 67: 3583-3590 (23 μm length); Walters, et al., 1997, SPIE, Proceedings Micro-Machining and Imaging 3009: 48 (26 μm length); and Schaeffer, et al., 1997, SPIE, Proceedings Micro-Machining and Imaging 3009: 48 (9 μm length).
A number of groups have made efforts to generate high performance cantilevers. Stowe, et al., 1997, Appl. Phys. Lett. 7(1): 288-290, describe ultrathin (60 nm) silicon cantilevers with force constants on the order of 10−6 and resonant frequencies in vacuum of 1.7 kHz. While the performance of these cantilevers in solution was not examined, the dimensions of the cantilevers are such that they have a predicted resonant frequency in water of about 200 Hz. This frequency is too low to be useful in most biological applications. Ried, et al., 1997, J. Microelectromechanical Sys. 6: 294-302, describe piezo resistive cantilevers with resonant frequencies of 6 MHz and spring constants of 2 N/m. However, these types of cantilevers have relatively poor detection sensitivity and are difficult to work with in solution, preventing them from being used productively in biological research. Such cantilevers are used instead in data storage applications (see, e.g., Mamin and Rugar, 1996, Appl. Phys. 79: 5644-5644) or in force-based magnetic resonant imaging (Rugar et al., 1994, Science 264: 1560-1563).
High performance cantilevers specifically for use in biological research have been described (Walters et al., 1996, Rev Sci Instrum 67:3583-3590; Viani, et al., 1999, Rev. Sci. Instrum. 70: 4300-4303). The best cantilevers thus far developed have resonant frequencies of 100-200 kHz in solution and spring constants of 0.1-0.2 N/m. However, improvements in these cantilevers are largely limited by the lithographic and thin film deposition methods used for their fabrication.
U.S. Pat. No. 6,016,693 discloses a method for making a smaller cantilever (e.g., 2-10 μm in length). The method comprises fabricating a “sacrificial cantilever” of SiO2 and depositing a layer of material which will form the final cantilever onto the sacrificial cantilever. The sacrificial cantilever is then etched away.
U.S. Pat. No. 5,666,190 discloses a compound cantilever for a scanning probe microscope which includes a bending portion and a vibrating portion. The vibrating portion has a lower mechanical resonant frequency than the bending portion. The cantilever is fabricated from two fused silicon oxide wafers.